Human embryonic stem cells (hESCs) are pluripotent cells that have the potential to be used for tissue engineering and regenerative medicine. Repairing nerve injury by differentiating hESCs into a neuronal lineage is one important application of hESCs. Biochemical and biological agents are widely used to induce hESC differentiation. However, it would be better if we could induce differentiation of hESCs without such agents because these factors are expensive and it is difficult to control the optimal concentrations for efficient differentiation with reduced side effects. Moreover, the mechanism of differentiation induced by these factors is still not fully understood. In this study, we present evidence that nanoscale ridge/groove pattern arrays alone can effectively and rapidly induce the differentiation of hESCs into a neuronal lineage without the use any differentiation-inducing agents. Using UV-assisted capillary force lithography, we constructed nanoscale ridge/groove pattern arrays with a dimension and alignment that were finely controlled over a large area. Human embryonic stem cells seeded onto the 350-nm ridge/groove pattern arrays differentiated into neuronal lineage after five days, in the absence differentiation-inducing agents. This nanoscale technique could be used for a new neuronal differentiation protocol of hESCs and may also be useful for nanostructured scaffolding for nerve injury repair.